&nbsp;&nbsp;&nbsp;&nbsp;In order to have a positional specificity, We make metal-deposited beads. Hemispherical part of polystyrene bead is covered with Cr,1/4 part of spere is covered with Au and the other part is polystyrene. First, deposit Au on the half of polystyrene beads. Second, deposit Cr on the half of polystyrene beads.

+

&nbsp;&nbsp;&nbsp;&nbsp;In order to have a positional specificity, We made metal-deposited beads. Hemispherical part of polystyrene bead was covered with Cr, 1/4 part of spere was covered with Au and the other part was polystyrene. First, deposited Au on the half of polystyrene beads. Second, deposited Cr on the half of polystyrene beads.

::'''DNA conjugation to polystyrene surface'''<br>

::'''DNA conjugation to polystyrene surface'''<br>

-

&nbsp;&nbsp;&nbsp;&nbsp;Carboxyl (COOH) microparticles can be used for covalent coupling of proteins by activating the carboxyl group with water-soluble carbodiimide. The carbodiimide reacts with the carboxyl group to create an active ester that is reactive toward primary amines on the DNA.<br>

+

&nbsp;&nbsp;&nbsp;&nbsp;Carboxyl (COOH) microparticles can be used for covalent coupling of proteins by activating the carboxyl group with water-soluble carbodiimide. The carbodiimide reacted with the carboxyl group to create an active ester that was reactive toward primary amines on the DNA.<br>

-

First, carboxyl group of polystyrene beads and EDAC react with each other. Next, Carboxyl group become active intermediate. Then DNA coupled to Polystyrene beads

+

&nbsp;&nbsp;&nbsp;&nbsp;First, carboxyl group of polystyrene beads and EDAC reacted with each other. Next, carboxyl group became active intermediate. Then DNA coupled to polystyrene beads.

::'''DNA conjugation to metal surface'''<br>

::'''DNA conjugation to metal surface'''<br>

-

&nbsp;&nbsp;&nbsp;&nbsp;When we mix thiol-modified DNA and Pt or Au, they will be self assembly. Thiol-modified DNA and Au or Pt are organized naturally. Using this characteristic, bond two kinds of DNA with Pt and bond another kind of DNA with Au.

+

&nbsp;&nbsp;&nbsp;&nbsp;When we mix thiol-modified DNA and Pt or Au, they will be self assembly. Thiol-modified DNA and Au or Pt are organized naturally. Using this characteristic, attached two kinds of DNA with Pt and bond another kind of DNA with Au.

::'''Catalyst conjugation'''

::'''Catalyst conjugation'''

&nbsp;&nbsp;&nbsp;&nbsp; Finally, we conjugated the catalyst to our rocket body by DNA hybridization. Complementary strand DNA had adhered to the corresponding parts of the rocket body, so it enabled to conjugate catalyst engines in that we designed these DNA were thermodynamically stable when they hybridized.

&nbsp;&nbsp;&nbsp;&nbsp; Finally, we conjugated the catalyst to our rocket body by DNA hybridization. Complementary strand DNA had adhered to the corresponding parts of the rocket body, so it enabled to conjugate catalyst engines in that we designed these DNA were thermodynamically stable when they hybridized.

&nbsp;&nbsp;&nbsp;&nbsp;We deposit gold on the polystyrene-bead to make gold hemisphered body.<br>

+

&nbsp;&nbsp;&nbsp;&nbsp;We deposit gold on the polystyrene-bead to make the body that have Au hemispherical area.<br><br>

-

&nbsp;&nbsp;&nbsp;&nbsp;2nd Deposition

+

2nd Deposition<br>

&nbsp;&nbsp;&nbsp;&nbsp;Next, We change the direction of beads, and deposit chromium on the body. Then we pick up polystyrene-bead, which are deposited by quarter gold and half chromium. By chromium capping, this "head part" is prevented to conjyugate DNA strands, so platinum only mounted at the rear.<br><br>

&nbsp;&nbsp;&nbsp;&nbsp;Next, We change the direction of beads, and deposit chromium on the body. Then we pick up polystyrene-bead, which are deposited by quarter gold and half chromium. By chromium capping, this "head part" is prevented to conjyugate DNA strands, so platinum only mounted at the rear.<br><br>

::::::::::>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#0-1.Selective_coating_of_the_body|Result]]

+

::::::::::>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#0.1._Selective_coating_of_the_body|Results]]

-

==0-2.DNA design==

+

==0.2. Design of linker DNA strands==

-

:{|

+

:&nbsp;&nbsp;&nbsp;&nbsp;We used DNA strands for bonding between a body of Biomolecular Rocket and platinum particles. DNA strands must be designed 4 DNA strands to hybridize at room temperature stably. DNA strands are categorize into two types, one type was a DNA strand for conjugation to polystyrene surface and its cDNA, and another type was a DNA strand for conjugation to gold surface and its cDNA. Each cDNA conjugated to platinum particles.<br>

-

|-

+

:&nbsp;&nbsp;&nbsp;&nbsp;In this section, we explain how we design DNA strands. There are three steps to design DNA strands, first step is selection of DNA strands , second step is adjustment of the number of bases and final step is design of cDNA strand. We used NUPACK<sup>[1]</sup> for these processes of DNA design. NUPACK is a software for the analysis and design of nucleic acid systems.<br>

:&nbsp;&nbsp;&nbsp;&nbsp;First, we chosed DNA strands from a treatise<sup>[2]</sup>. In this step, we analyzed whether DNA could easily hybridize at room temperature stably. Then we selected two DNA strands. Secondly, we adjusted number of bases in order to make easily dissociated when they introduced photo-switching systems. Finally, we design c of each cDNA strands, and modify amino group and thiol group as necessary. <br>

-

&nbsp;&nbsp;&nbsp;&nbsp;We use DNA strands for bonding between a body of Biomolecular Rocket and platinum particles. DNA strands must be designed 4 DNA strands to hybridize at room temperature stably. DNA strands are categorize into two types, one type is a DNA strand for conjugation to polystyrene surface and its cDNA, and another type is a DNA strand for conjugation to gold surface and its cDNA. Each cDNA conjugated to platinum particles.<br>

+

:Reference

-

&nbsp;&nbsp;&nbsp;&nbsp;In this section, we explain how we design DNA strands. There are three steps to design DNA strands, first step is selection of DNA strands , second step is adjustment of the number of bases and final step is design of cDNA strand. We use NUPACK*1 for these processes of DNA design. NUPACK is a software for the analysis and design of nucleic acid systems.

:&nbsp;&nbsp;&nbsp;&nbsp;First, we chose DNA strands from a treatise*2. In this step, we analyze whether DNA can easily hybridize at room temperature stably. Then we select two DNA strands. Secondly, we adjust number of bases in order to make easily dissociate when they introduced photo-switching systems. Finally, we design c of each cDNA strands, and modify amino group and thiol group as necessary. <br>

+

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#0.2._Design_of_linker_DNA_strands|Results]]

&nbsp;&nbsp;&nbsp;&nbsp;'''We bond polystyrene-beads with DNA using EDAC.''' EDAC is known as water-soluble carbodiimide (WSC) and is used as coupling agent to form amide bond. EDAC crosslinks nucleic acids to the surfaces, which have carboxy group. In this project, we used this method to conjugate amino-modified DNA onto carboxylated polystyrene-beads. EDAC reacts with carboxylated polystyrene-beads to form active intermediate on the body. Then it reacts to amino-modified DNA. The beads are coupled by nucleic acid. We divide the body into three parts including polystyrene surface, so it is possible to connect each DNA with position classification. By using this method, we can connect platinum to aircraft body part of polystyrene in a region-specific manner.

+

&nbsp;&nbsp;&nbsp;&nbsp;'''We bond polystyrene-beads with DNA using EDAC.''' EDAC is known as water-soluble carbodiimide (WSC) and is used as coupling agent to form amide bond. EDAC crosslinks nucleic acids to the surfaces, which have carboxy group. In this project, we used this method to conjugate amino-modified DNA onto carboxylated polystyrene-beads. EDAC reacted with carboxylated polystyrene-beads to form active intermediate on the body. Then it reacted to amino-modified DNA. The beads are coupled by nucleic acid. We divide the body into three parts including polystyrene surface, so it is possible to connect each DNA with position classification. By using this method, we were able to connect platinum to aircraft body part of polystyrene in a region-specific manner.

|}

|}

-

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#0-3-1.Selective_conjugation_of_DNA_to_polystyrene_surface_area|Result]]

+

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#0.3.1._Selective_conjugation_of_DNA_to_polystyrene_surface_area|Results]]

&nbsp;&nbsp;&nbsp;&nbsp;'''Self-assembled monolayers (SAM)''' of molecules are molecular assemblies formed spontaneously on substrate surfaces by chemical adsorption. This chemical reactions can be organized into large ordered domains and it is also possible to cover the substrate with functional end group that has some modification. In addition, few steps and experimental technique are required. SAMs are created by adsorption of head groups onto a substrate followed by a slow organization of tail groups.<br><br>

+

&nbsp;&nbsp;&nbsp;&nbsp;'''Self-assembled monolayers (SAM)<sup>[1],[2]</sup>''' of molecules are molecular assemblies formed spontaneously on substrate surfaces by chemical adsorption. This chemical reactions can be organized into large ordered domains and it is also possible to cover the substrate with functional end group that has some modification. In addition, few steps and experimental technique are required. SAMs are created by adsorption of head groups onto a substrate followed by a slow organization of tail groups.<br><br>

&nbsp;&nbsp;&nbsp;First, head groups are absorbed onto substrate over . In this phase, the surface of substrate is patterned with spots by dsorbeate molecules rather than beautiful membranes.

&nbsp;&nbsp;&nbsp;First, head groups are absorbed onto substrate over . In this phase, the surface of substrate is patterned with spots by dsorbeate molecules rather than beautiful membranes.

|-

|-

Line 69:

Line 66:

|

|

SAM conjugation onto gold<br>

SAM conjugation onto gold<br>

-

&nbsp;&nbsp;&nbsp;&nbsp;Thiolated compound and gold substrate are one of the most famous research of SAMs. Thiol modified DNA is conjugate to gold particles by this chemical reaction. Initially, substrate gold are cleaned by water or acid-base buffer in order to washout organic impurities. Gold particles are dissolved in 10mM phosphate buffer pH8, 10μM thiol modified DNA. Then in addition to NaCl, intended to raise the concentration of NaCl every 2 hours after 24 hours of incubation for 6 hours, then incubate more 16 hoursraise up to 0.7M salt concentration '''(Salt aging process)'''.Last,clean up again by proper buffer and dry. This reaction is also use ​​with platinum In order to accomplish our project ,we need platinum-DNA and gold-DNA conjugation, so we use thiol modified DNA.

+

&nbsp;&nbsp;&nbsp;&nbsp;Thiolated compound and gold substrate are one of the most famous research of SAMs<sup>[3]</sup>. Thiol modified DNA was conjugate to gold particles by this chemical reaction. Initially, substrate gold were cleaned by water or acid-base buffer in order to washout organic impurities. Gold particles were dissolved in 10mM phosphate buffer pH8, 10μM thiol modified DNA. Then in addition to NaCl, intended to raise the concentration of NaCl every 2 hours after 24 hours of incubation for 6 hours, then incubate more 16 hoursraise up to 0.7M salt concentration '''(Salt aging process)'''.Last,clean up again by proper buffer and dry. This reaction was also used ​​with platinum.

|-

|-

|}

|}

-

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#0-3-2.Selective_conjugation_of_DNA_to_metal_surface_area|Result]]

&nbsp;&nbsp;&nbsp;&nbsp;'''We conjugated the catalyst to our rocket body by DNA hybridization.''' cDNA had adhered to the corresponding parts of the rocket body, so it enabled to conjugate catalyst engines in that we designed these DNA were thermodynamically stable when they hybridized.<br><br>

+

&nbsp;&nbsp;&nbsp;&nbsp;'''We attached the catalyst to our rocket body with DNA hybridization.''' cDNA had adhered to the corresponding parts of the rocket body, so it enabled to attach catalyst engines in that we designed these DNA were thermodynamically stable when they hybridized.<br><br>

-

&nbsp;&nbsp;&nbsp;&nbsp;First, we prepared DNA-conjugated 10 μm sized polystyrene beads which were deposited 1/4 Au and 1/2 Cr. At the same time we prepared DNA-conjugated 0.15~0.40 μm sized platinum particles. Then, mixed these materials in 3×SSC buffer at 90℃, and cooled to room temperature slowly over time. Namely, annealing DNA by raising temperature at the first time, next make it easy to hybridize each other by downing to room temperature.

+

&nbsp;&nbsp;&nbsp;&nbsp;First, we prepared DNA-conjugated 10 μm sized polystyrene beads which were deposited 1/4 Au and 1/2 Cr. At the same time we prepared DNA-conjugated 0.15-0.40 μm sized platinum particles. Then, mixed these materials in 3×SSC buffer at 90 °C, and cooled to room temperature slowly over time. Namely, annealing DNA by raising temperature at the first time, next make it easy to hybridize each other by downing to room temperature.

|}

|}

-

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#0-4.Catalyst_conjugation_by_DNA_hybridization|Result]]

+

::::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#0.4._Catalyst_attachment_with_DNA_hybridization|Results]]

:&nbsp;&nbsp;&nbsp;&nbsp;H<sub>2</sub>O<sub>2</sub> has strong corrosion. There was a risk that DNA strand is denatured by H<sub>2</sub>O<sub>2</sub>. So, we had to ensure that the hybridization of DNA is not suffer from H<sub>2</sub>O<sub>2</sub>. We guess that if DNA had been destroyed, then molecular mass would decrease in that DNA can't hybridize. Measure the relative magnitude of the molecular weight by electrophoresis, we are able to examaine whether DNA can hybridize or not.<br>

+

:&nbsp;&nbsp;&nbsp;&nbsp;H<sub>2</sub>O<sub>2</sub> has strong corrosion. There was a risk that DNA strand is denatured by H<sub>2</sub>O<sub>2</sub><sup>[1]</sup>. So, we had to ensure that the hybridization of DNA is not suffer from H<sub>2</sub>O<sub>2</sub>. We guessed that if DNA had been destroyed, then molecular mass would decrease in that DNA can't hybridize. Visualizing the relative magnitude of the molecular weight by electrophoresis, we are able to examine whether DNA can hybridize or not.<br>

-

:&nbsp;&nbsp;&nbsp;&nbsp;This time, we have to examine the state of the DNA in the time we need to steer rocket, set the time to soak and concentration of H<small>2</small>O<small>2</small>. We used PAGE electrophoresis to ascertain the stability of DNA duplex in thin H₂O₂ solution 1%~5%. PAGE electrophoresis shows the difference of molecular weight that comes from denaturetion or hybridization in the form of bands.

+

:&nbsp;&nbsp;&nbsp;&nbsp;This time, we have to examine the state of the DNA in the time we need to steer rocket, set the time to soak and concentration of H<small>2</small>O<small>2</small>. We used PAGE electrophoresis to ascertain the stability of DNA duplex in thin H₂O₂ solution 1%-5%. PAGE electrophoresis shows the difference of molecular weight that comes from dissociation or hybridization in the form of bands.<br><br>

-

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#1-1.DNA_hybridization_in_solution_of_H2O2|Result]]

==1-2.Verification of platinum hemisphere behavior in solution of H<sub>2</sub>O<sub>2</sub>==

+

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#1.1._DNA_hybridization_in_solution_of_H2O2|Results]]

-

:&nbsp;&nbsp;&nbsp;&nbsp;The driving force of Biomolecular Rocket is catalytic engine of platinum. This catalytic engine become more powerful, increasing the surface of platinum. Biomolecular Rocket has numerous platinum particles to increase the surface of platinum. In this experiment, we deposit chromium to 1µm beads which are covered platinum and we make half platinum beads. In addition, we observe a behavior of the beads in 3% H<sub>2</sub>O<sub>2</sub> solution. From this experiment, we are able to investigate whether Surface area of platinum is related to the speed.<br>

+

-

[[Image:Make platinum hemisphere.png|center|200px]]

+

-

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#1-2.Verification_of_platinum_hemisphere_behavior_in_solution_of_H2O2|Result]]

+

-

==1-3.Verification of catalase hemisphere behavior in solution of H<small>2</small>O<small>2</small>==

+

==1.2. Power supply for rail-free movement by using catalase catalytic engine==

&nbsp;&nbsp;&nbsp;&nbsp;For supplying power of Biomolecular Rocket, we experiment with another catalytic engine. Catalase is known as a enzyme that catalyzes the hydrogen peroxide solution. And we expect that this enzyme will be new catalytic engine of the Biomolecular Rocket because a enzyme activity of this enzyme is 100 000 times stronger than that of platinum. In this experiment, we deposit chromium for half surface of polystyrene beads and catalase conjugated onto polystyrene side. In addition, we observe a behavior of the beads in diluted H<small>2</small>O<small>2</small> solution.<br>

+

&nbsp;&nbsp;&nbsp;&nbsp;For supplying power of Biomolecular Rocket, we experimented with another catalytic engine<sup>[1],[2],[3]</sup>. Catalase is known as a enzyme that catalyzes the hydrogen peroxide solution. And we expected that this enzyme will be new catalytic engine of the Biomolecular Rocket because a enzyme activity of this enzyme is 100,000 times stronger than that of platinum. In this experiment, we deposited Au for half surface of polystyrene beads and catalase conjugated onto polystyrene side. In addition, we observed a behavior of the beads in diluted H<small>2</small>O<small>2</small> solution.<br>

[[Image:Make catalase rocket.png|center|350px]]

[[Image:Make catalase rocket.png|center|350px]]

|}

|}

-

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#1-3.Verification_of_catalase_hemisphere_behavior_in_solution_of_H2O2|Result]]

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#1.2._Power_supply_for_rail-free_movement_by_using_catalase_catalytic_engine|Results]]

+

+

==1.3. Power supply for rail-free movement by using platinum catalytic engine==

+

:&nbsp;&nbsp;&nbsp;&nbsp;The driving force of Biomolecular Rocket is catalytic engine of platinum. This catalytic engine became more powerful<sup>[1],[2]</sup>, increasing the surface of platinum. Biomolecular Rocket had numerous platinum particles to increase the surface of platinum. In this experiment, we deposited chromium to 1µm beads which are covered platinum and we make half platinum beads<sup>[3]</sup>. Then, we observed a behavior of the beads in 3% H<sub>2</sub>O<sub>2</sub> solution. From this experiment, we are able to investigate whether Surface area of platinum was related to the speed.<br>

:&nbsp;&nbsp;&nbsp;&nbsp;We incorporated 4 azobenzene units into photoresponsive DNA<sup>[1]</sup> strand so that we can quickly dissociated DNA duplex. We called it as Photoresponsive DNA sequence S.[5'-AATXACXCCXAGXCC-3' (X=Azobenzene)]

:&nbsp;&nbsp;&nbsp;&nbsp;The value of Abs is proportional to the concentration of corresponding materials. If photoresponsive DNA formed duplex, the concentration of ssDNA decrease, onthe other hand the concentration of dsDNA increase. Reserchin these corresponding values, we could determine whether they formed duplex or not.

+

:&nbsp;&nbsp;&nbsp;&nbsp;The value of Abs is proportional to the concentration of corresponding materials. If photoresponsive DNA formed duplex, the concentration of ssDNA decrease, onthe other hand the concentration of dsDNA increase. Reserchin these corresponding values, we could determine whether they formed duplex or not.<br>

-

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#3-1.Design_of_photoresponsive_DNA|Result]]

&nbsp;&nbsp;&nbsp;&nbsp;The duplex-forming activities of DNA can be photomodulated by incorporation of an azobenzene unit. The duplex is dissociated on isomerizing the trans-azobenzene to the cis form by irradiation with UV light. The duplex is formed again when the cis-azobenzene is converted to the transazobenzene by irradiation with visible light. <br>

+

&nbsp;&nbsp;&nbsp;&nbsp;The duplex-forming activities of DNA can be photomodulated by incorporation of an azobenzene unit<sup>[1]</sup>. The duplex is dissociated on isomerizing the trans-azobenzene to the cis form by irradiation with UV light. The duplex is formed again when the cis-azobenzene is converted to the transazobenzene by irradiation with visible light. <br>

&nbsp;&nbsp;&nbsp;&nbsp;We inserted this function into a bead-body and add the photo-switching function to the body.In brief, so that we can detached the engines by irradiation with UV light, we attached a part of engines to the body with DNA duplex incorporating azobenzene unit.

&nbsp;&nbsp;&nbsp;&nbsp;We inserted this function into a bead-body and add the photo-switching function to the body.In brief, so that we can detached the engines by irradiation with UV light, we attached a part of engines to the body with DNA duplex incorporating azobenzene unit.

|}

|}

-

:&nbsp;&nbsp;&nbsp;&nbsp;We measured DNA absorbance and ascertain the duplex-forming and duplex-dissociation activities of photoswitcable DNA strands. As the ordered regions of stacked base pairs in the DNA duplex are dissociated, the UV absorbance increases. This difference in absorbance between the duplex and single strand state is the result of nearest neighbor base pair interactions. In other words, when the DNA is in the duplex state, interactions between base pairs decrease the UV absorbance relative to single strands. When the DNA is in the single strand state the interactions are much weaker,due to the decreased proximity, and the UV absorbance is higher than the duplex state. About this time, we analyze the rerationship between the time of dissociation photo-switcable DNA and the strength of the UV light.

+

:&nbsp;&nbsp;&nbsp;&nbsp;We measured DNA absorbance and ascertain the duplex-forming and duplex-dissociation activities of photoswitcable DNA strands. As the ordered regions of stacked base pairs in the DNA duplex are dissociated, the UV absorbance increases. This difference in absorbance between the duplex and single strand state is the result of nearest neighbor base pair interactions. In other words, when the DNA is in the duplex state, interactions between base pairs decrease the UV absorbance relative to single strands. When the DNA is in the single strand state the interactions are much weaker,due to the decreased proximity, and the UV absorbance is higher than the duplex state. About this time, we analyze the rerationship between the time of dissociation photo-switcable DNA and the strength of the UV light. <br>

-

::::::::::>>back to [[Biomod/2012/Titech/Nano-Jugglers/Results#3-2.Dissociation_of_photoresponsive_DNA_by_UV-light_irradiation|Result]]

Current revision

0.Construction of Biomolecular Rocket

Selective coating by vapor deposition

In order to have a positional specificity, We made metal-deposited beads. Hemispherical part of polystyrene bead was covered with Cr, 1/4 part of spere was covered with Au and the other part was polystyrene. First, deposited Au on the half of polystyrene beads. Second, deposited Cr on the half of polystyrene beads.

DNA conjugation to polystyrene surface

Carboxyl (COOH) microparticles can be used for covalent coupling of proteins by activating the carboxyl group with water-soluble carbodiimide. The carbodiimide reacted with the carboxyl group to create an active ester that was reactive toward primary amines on the DNA.
First, carboxyl group of polystyrene beads and EDAC reacted with each other. Next, carboxyl group became active intermediate. Then DNA coupled to polystyrene beads.

DNA conjugation to metal surface

When we mix thiol-modified DNA and Pt or Au, they will be self assembly. Thiol-modified DNA and Au or Pt are organized naturally. Using this characteristic, attached two kinds of DNA with Pt and bond another kind of DNA with Au.

Catalyst conjugation

Finally, we conjugated the catalyst to our rocket body by DNA hybridization. Complementary strand DNA had adhered to the corresponding parts of the rocket body, so it enabled to conjugate catalyst engines in that we designed these DNA were thermodynamically stable when they hybridized.
we build our rocket in this way.

0.1. Selective coating of the body

1st Deposition
We deposit gold on the polystyrene-bead to make the body that have Au hemispherical area.

2nd Deposition
Next, We change the direction of beads, and deposit chromium on the body. Then we pick up polystyrene-bead, which are deposited by quarter gold and half chromium. By chromium capping, this "head part" is prevented to conjyugate DNA strands, so platinum only mounted at the rear.

0.2. Design of linker DNA strands

We used DNA strands for bonding between a body of Biomolecular Rocket and platinum particles. DNA strands must be designed 4 DNA strands to hybridize at room temperature stably. DNA strands are categorize into two types, one type was a DNA strand for conjugation to polystyrene surface and its cDNA, and another type was a DNA strand for conjugation to gold surface and its cDNA. Each cDNA conjugated to platinum particles.

In this section, we explain how we design DNA strands. There are three steps to design DNA strands, first step is selection of DNA strands , second step is adjustment of the number of bases and final step is design of cDNA strand. We used NUPACK[1] for these processes of DNA design. NUPACK is a software for the analysis and design of nucleic acid systems.

First, we chosed DNA strands from a treatise[2]. In this step, we analyzed whether DNA could easily hybridize at room temperature stably. Then we selected two DNA strands. Secondly, we adjusted number of bases in order to make easily dissociated when they introduced photo-switching systems. Finally, we design c of each cDNA strands, and modify amino group and thiol group as necessary.

0-3.DNA conjugation

0.3.1. Selective conjugation of DNA to polystyrene surface area

We bond polystyrene-beads with DNA using EDAC. EDAC is known as water-soluble carbodiimide (WSC) and is used as coupling agent to form amide bond. EDAC crosslinks nucleic acids to the surfaces, which have carboxy group. In this project, we used this method to conjugate amino-modified DNA onto carboxylated polystyrene-beads. EDAC reacted with carboxylated polystyrene-beads to form active intermediate on the body. Then it reacted to amino-modified DNA. The beads are coupled by nucleic acid. We divide the body into three parts including polystyrene surface, so it is possible to connect each DNA with position classification. By using this method, we were able to connect platinum to aircraft body part of polystyrene in a region-specific manner.

0.3.2. Selective conjugation of DNA to metal surface area

Self-assembled monolayers (SAM)[1],[2] of molecules are molecular assemblies formed spontaneously on substrate surfaces by chemical adsorption. This chemical reactions can be organized into large ordered domains and it is also possible to cover the substrate with functional end group that has some modification. In addition, few steps and experimental technique are required. SAMs are created by adsorption of head groups onto a substrate followed by a slow organization of tail groups.

First, head groups are absorbed onto substrate over . In this phase, the surface of substrate is patterned with spots by dsorbeate molecules rather than beautiful membranes.

Over the period of disordering form, the head groups assemble together on the substrate, while the tail groups assemble far from the substrate. Areas of concentrated molecules nucleate and grow until the surface of the substrate is covered in a single monolayer.

SAM conjugation onto gold
Thiolated compound and gold substrate are one of the most famous research of SAMs[3]. Thiol modified DNA was conjugate to gold particles by this chemical reaction. Initially, substrate gold were cleaned by water or acid-base buffer in order to washout organic impurities. Gold particles were dissolved in 10mM phosphate buffer pH8, 10μM thiol modified DNA. Then in addition to NaCl, intended to raise the concentration of NaCl every 2 hours after 24 hours of incubation for 6 hours, then incubate more 16 hoursraise up to 0.7M salt concentration (Salt aging process).Last,clean up again by proper buffer and dry. This reaction was also used ​​with platinum.

0.4. Catalyst attachment with DNA hybridization

We attached the catalyst to our rocket body with DNA hybridization. cDNA had adhered to the corresponding parts of the rocket body, so it enabled to attach catalyst engines in that we designed these DNA were thermodynamically stable when they hybridized.

First, we prepared DNA-conjugated 10 μm sized polystyrene beads which were deposited 1/4 Au and 1/2 Cr. At the same time we prepared DNA-conjugated 0.15-0.40 μm sized platinum particles. Then, mixed these materials in 3×SSC buffer at 90 °C, and cooled to room temperature slowly over time. Namely, annealing DNA by raising temperature at the first time, next make it easy to hybridize each other by downing to room temperature.

1.1. DNA hybridization in solution of H2O2

H2O2 has strong corrosion. There was a risk that DNA strand is denatured by H2O2[1]. So, we had to ensure that the hybridization of DNA is not suffer from H2O2. We guessed that if DNA had been destroyed, then molecular mass would decrease in that DNA can't hybridize. Visualizing the relative magnitude of the molecular weight by electrophoresis, we are able to examine whether DNA can hybridize or not.

This time, we have to examine the state of the DNA in the time we need to steer rocket, set the time to soak and concentration of H2O2. We used PAGE electrophoresis to ascertain the stability of DNA duplex in thin H₂O₂ solution 1%-5%. PAGE electrophoresis shows the difference of molecular weight that comes from dissociation or hybridization in the form of bands.

1.2. Power supply for rail-free movement by using catalase catalytic engine

For supplying power of Biomolecular Rocket, we experimented with another catalytic engine[1],[2],[3]. Catalase is known as a enzyme that catalyzes the hydrogen peroxide solution. And we expected that this enzyme will be new catalytic engine of the Biomolecular Rocket because a enzyme activity of this enzyme is 100,000 times stronger than that of platinum. In this experiment, we deposited Au for half surface of polystyrene beads and catalase conjugated onto polystyrene side. In addition, we observed a behavior of the beads in diluted H2O2 solution.

1.3. Power supply for rail-free movement by using platinum catalytic engine

The driving force of Biomolecular Rocket is catalytic engine of platinum. This catalytic engine became more powerful[1],[2], increasing the surface of platinum. Biomolecular Rocket had numerous platinum particles to increase the surface of platinum. In this experiment, we deposited chromium to 1µm beads which are covered platinum and we make half platinum beads[3]. Then, we observed a behavior of the beads in 3% H2O2 solution. From this experiment, we are able to investigate whether Surface area of platinum was related to the speed.

3.1. Design of photoresponsive DNA

We incorporated 4 azobenzene units into photoresponsive DNA[1] strand so that we can quickly dissociated DNA duplex. We called it as Photoresponsive DNA sequence S.[5'-AATXACXCCXAGXCC-3' (X=Azobenzene)]

The value of Abs is proportional to the concentration of corresponding materials. If photoresponsive DNA formed duplex, the concentration of ssDNA decrease, onthe other hand the concentration of dsDNA increase. Reserchin these corresponding values, we could determine whether they formed duplex or not.

3.2. Directional control with photo-switchable DNA system

The duplex-forming activities of DNA can be photomodulated by incorporation of an azobenzene unit[1]. The duplex is dissociated on isomerizing the trans-azobenzene to the cis form by irradiation with UV light. The duplex is formed again when the cis-azobenzene is converted to the transazobenzene by irradiation with visible light.
We inserted this function into a bead-body and add the photo-switching function to the body.In brief, so that we can detached the engines by irradiation with UV light, we attached a part of engines to the body with DNA duplex incorporating azobenzene unit.

We measured DNA absorbance and ascertain the duplex-forming and duplex-dissociation activities of photoswitcable DNA strands. As the ordered regions of stacked base pairs in the DNA duplex are dissociated, the UV absorbance increases. This difference in absorbance between the duplex and single strand state is the result of nearest neighbor base pair interactions. In other words, when the DNA is in the duplex state, interactions between base pairs decrease the UV absorbance relative to single strands. When the DNA is in the single strand state the interactions are much weaker,due to the decreased proximity, and the UV absorbance is higher than the duplex state. About this time, we analyze the rerationship between the time of dissociation photo-switcable DNA and the strength of the UV light.